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Colonization and infection of soybean cotyledons by Agrobacterium tumefaciens and subsequent elimination of bacteria from cotyledons were monitored using bacteria expressing green fluorescent protein (GFP). GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells. GFP was first detected 7 h following inoculation of the cotyledon. By 36 h, GFP expression was very intense, and was limited to the adaxial surface of the cotyledon. Expression of GFP also served as a useful indicator of successful elimination of the bacterium from plant tissue following antibiotic treatment.